EphrinA2 receptor (EphA2) is an invasion and intracellular signaling receptor for Chlamydia trachomatis.

The obligate intracellular bacterium Chlamydia trachomatis invades into host cells to replicate inside a membrane-bound vacuole called inclusion. Multiple different host proteins are recruited to the inclusion and are functionally modulated to support chlamydial development. Invaded and replicating Chlamydia induces a long-lasting activation of the PI3 kinase signaling pathway that is required for efficient replication. We identified the cell surface tyrosine kinase EphrinA2 receptor (EphA2) as a chlamydial adherence and invasion receptor that induces PI3 kinase (PI3K) activation, promoting chlamydial replication. Interfering with binding of C. trachomatis serovar L2 (Ctr) to EphA2, downregulation of EphA2 expression or inhibition of EphA2 activity significantly reduced Ctr infection. Ctr interacts with and activates EphA2 on the cell surface resulting in Ctr and receptor internalization. During chlamydial replication, EphA2 remains active accumulating around the inclusion and interacts with the p85 regulatory subunit of PI3K to support the activation of the PI3K/Akt signaling pathway that is required for normal chlamydial development. Overexpression of full length EphA2, but not the mutant form lacking the intracellular cytoplasmic domain, enhanced PI3K activation and Ctr infection. Despite the depletion of EphA2 from the cell surface, Ctr infection induces upregulation of EphA2 through the activation of the ERK pathway, which keeps the infected cell in an apoptosis-resistant state. The significance of EphA2 as an entry and intracellular signaling receptor was also observed with the urogenital C. trachomatis-serovar D. Our findings provide the first evidence for a host cell surface receptor that is exploited for invasion as well as for receptor-mediated intracellular signaling to facilitate chlamydial replication. In addition, the engagement of a cell surface receptor at the inclusion membrane is a new mechanism by which Chlamydia subverts the host cell and induces apoptosis resistance.

Klebsiella pneumoniae targets an EGF receptor-dependent pathway to subvert inflammation.

The NF-κB transcriptional factor plays a key role governing the activation of immune responses. Klebsiella pneumoniae is an important cause of community-acquired and nosocomial pneumonia. Evidence indicates that K. pneumoniae infections are characterized by lacking an early inflammatory response. Recently, we have demonstrated that Klebsiella antagonizes the activation of NF-κB via the deubiquitinase CYLD. In this work, by applying a high-throughput siRNA gain-of-function screen interrogating the human kinome, we identified 17 kinases that when targeted by siRNA restored IL-1ß-dependent NF-κB translocation in infected cells. Further characterization revealed that K. pneumoniae activates an EGF receptor (EGFR)-phosphatidylinositol 3-OH kinase (PI3K)-AKT-PAK4-ERK-GSK3ß signalling pathway to attenuate the cytokine-dependent nuclear translocation of NF-κB. Our data also revealed that CYLD is a downstream effector of K. pneumoniae-induced EGFR-PI3K-AKT-PAK4-ERK-GSK3ß signalling pathway. Our efforts to identify the bacterial factor(s)responsible for EGFR activation demonstrate that a capsule (CPS) mutant did not activate EGFR hence suggesting that CPS could mediate the activation of EGFR. Supporting this notion, purified CPS did activate EGFR as well as the EGFR-dependent PI3K-AKT-PAK4-ERK-GSK3ß signalling pathway. CPS-mediated EGFR activation was dependent on a TLR4-MyD88-c-SRC-dependent pathway. Several promising drugs have been developed to antagonize this cascade. We propose that agents targeting this signalling pathway might provide selective alternatives for the management of K. pneumoniae pneumonias.

Signal peptidase complex subunit 1 is an essential Zika virus host factor in placental trophoblasts.

Zika is a major teratogenic virus that can be transmitted from pregnant women to the fetus via the transplacental route. At present, no specific vaccines or treatments are available. Large-scale functional genomics approaches for the analysis of host cell function in infection greatly improve the understanding of molecular infection processes and advance the discovery of antiviral targets. We conducted a pooled CRISPR/Cas9 screen to explore trophoblast function upon Zika infection. The identified Zika virus host factors enrich in the ER membrane complex and the signal peptide processing pathway. Finally, we demonstrate that signal peptidase complex subunit 1 (SPCS1) is crucial for virus replication in trophoblasts.

Discovery of Zika virus host dependency factors in trophoblasts using CRISPR/Cas9 screening.

Emerging mosquito-borne RNA viruses cause massive health complications worldwide. The Zika virus (ZIKV), in particular, has spread dramatically since 2007 and has provoked epidemics in the Americas and the South Pacific. The lack of antiviral therapy and vaccination has focused research on the investigation of ZIKV-host interactions, in order to understand underlying molecular infection mechanisms. We have established an approach for the analysis of ZIKV host dependency factors in a human trophoblast cell line and applied genome-wide CRISPR/Cas9 knockout mutagenesis. The presented method is especially of value for the identification of factors that are essential for placental infection with the potential to serve as targets for antiviral treatment.

Modulation of Host Cell Metabolism by Chlamydia trachomatis.

Propagation of the intracellular bacterial pathogen Chlamydia trachomatis is strictly bound to its host cells. The bacterium has evolved by minimizing its genome size at the cost of being completely dependent on its host. Many of the vital nutrients are synthesized only by the host, and this has complex implications. Recent advances in loss-of-function analyses and the metabolomics of human infected versus noninfected cells have provided comprehensive insight into the molecular changes that host cells undergo during the stage of infection. Strikingly, infected cells acquire a stage of high metabolic activity, featuring distinct aspects of the Warburg effect, a condition originally assigned to cancer cells. This condition is characterized by aerobic glycolysis and an accumulation of certain metabolites, altogether promoting the synthesis of crucial cellular building blocks, such as nucleotides required for DNA and RNA synthesis. The altered metabolic program enables tumor cells to rapidly proliferate as well as C. trachomatis-infected cells to feed their occupants and still survive. This program is largely orchestrated by a central control board, the tumor suppressor protein p53. Its downregulation in C. trachomatis-infected cells or mutation in cancer cells not only alters the metabolic state of cells but also conveys the prevention of programmed cell death involving mitochondrial pathways. While this points toward common features in the metabolic reprogramming of infected and rapidly proliferating cells, it also forwards novel treatment options against chronic intracellular infections involving well-characterized host cell targets and established drugs.

Combined Human Genome-wide RNAi and Metabolite Analyses Identify IMPDH as a Host-Directed Target against Chlamydia Infection.

Chlamydia trachomatis (Ctr) accounts for >130 million human infections annually. Since chronic Ctr infections are extremely difficult to treat, there is an urgent need for more effective therapeutics. As an obligate intracellular bacterium, Ctr strictly depends on the functional contribution of the host cell. Here, we combined a human genome-wide RNA interference screen with metabolic profiling to obtain detailed understanding of changes in the infected cell and identify druggable pathways essential for Ctr growth. We demonstrate that Ctr shifts the host metabolism toward aerobic glycolysis, consistent with increased biomass requirement. We identify key regulator complexes of glucose and nucleotide metabolism that govern Ctr infection processes. Pharmacological targeting of inosine-5'-monophosphate dehydrogenase (IMPDH), the rate-limiting enzyme in guanine nucleotide biosynthesis, efficiently inhibits Ctr growth both in vitro and in vivo. These results highlight the potency of genome-scale functional screening for the discovery of drug targets against bacterial infections.

ALPK1- and TIFA-Dependent Innate Immune Response Triggered by the Helicobacter pylori Type IV Secretion System.

Activation of transcription factor NF-κB is a hallmark of infection with the gastric pathogen Helicobacter pylori, associated with inflammation and carcinogenesis. Genome-wide RNAi screening revealed numerous host factors involved in H. pylori-, but not IL-1ß- and TNF-α-dependent NF-κB regulation. Pathway analysis including CRISPR/Cas9-knockout and recombinant protein technology, immunofluorescence microscopy, immunoblotting, mass spectrometry, and mutant H. pylori strains identified the H. pylori metabolite D-glycero-ß-D-manno-heptose 1,7-bisphosphate (ßHBP) as a cagPAI type IV secretion system (T4SS)-dependent effector of NF-κB activation in infected cells. Upon pathogen-host cell contact, TIFA forms large complexes (TIFAsomes) including interacting host factors, such as TRAF2. NF-κB activation, TIFA phosphorylation, and TIFAsome formation depend on a functional ALPK1 kinase, highlighting the ALPK1-TIFA axis as a core innate immune pathway. ALPK1-TIFA-mediated NF-κB activation was independent of CagA protein translocation, indicating that CagA translocation and HBP delivery to host cells are distinct features of the pathogen's T4SS.

Chlamydia infection depends on a functional MDM2-p53 axis.

Chlamydia, a major human bacterial pathogen, assumes effective strategies to protect infected cells against death-inducing stimuli, thereby ensuring completion of its developmental cycle. Paired with its capacity to cause extensive host DNA damage, this poses a potential risk of malignant transformation, consistent with circumstantial epidemiological evidence. Here we reveal a dramatic depletion of p53, a tumor suppressor deregulated in many cancers, during Chlamydia infection. Using biochemical approaches and live imaging of individual cells, we demonstrate that p53 diminution requires phosphorylation of Murine Double Minute 2 (MDM2; a ubiquitin ligase) and subsequent interaction of phospho-MDM2 with p53 before induced proteasomal degradation. Strikingly, inhibition of the p53-MDM2 interaction is sufficient to disrupt intracellular development of Chlamydia and interferes with the pathogen's anti-apoptotic effect on host cells. This highlights the dependency of the pathogen on a functional MDM2-p53 axis and lends support to a potentially pro-carcinogenic effect of chlamydial infection.

Critical role of methylglyoxal and AGE in mycobacteria-induced macrophage apoptosis and activation.

Apoptosis and activation of macrophages play an important role in the host response to mycobacterial infection involving TNF-alpha as a critical autocrine mediator. The underlying mechanisms are still ill-defined. Here, we demonstrate elevated levels of methylglyoxal (MG), a small and reactive molecule that is usually a physiological product of various metabolic pathways, and advanced glycation end products (AGE) during mycobacterial infection of macrophages, leading to apoptosis and activation of macrophages. Moreover, we demonstrate abundant AGE in pulmonary lesions of tuberculosis (TB) patients. Global gene expression profiling of MG-treated macrophages revealed a diverse spectrum of functions induced by MG, including apoptosis and immune response. Our results not only provide first evidence for the involvement of MG and AGE in TB, but also form a basis for novel intervention strategies against infectious diseases in which MG and AGE play critical roles.

Detection of Chlamydophila pneumoniae in the bone marrow of two patients with unexplained chronic anaemia.

Anaemia of chronic disease (ACD) is a common finding involving iron deficiency and signs of inflammation. Here, we report on two patients with ACD where a persistent infection with Chlamydophila (Chlamydia) pneumoniae (CP) was detected in bone marrow (BM) biopsies. Infection was suspected by routine cytology and confirmed by immunofluorescence, electron microscopy, polymerase chain reaction (PCR) including different primer sets and laboratories and sequencing of the PCR product. This is a first report of chlamydial presence in the BM of anaemic patients. The cases are presented because persistent chlamydial infection may contribute more frequently to chronic refractory anaemia than previously suspected.